B and Supplementary Fig. 2b). Electron density was clearly interpretable for
B and Supplementary Fig. 2b). Electron density was clearly interpretable for the SSM and `RBD’5 but not for amino acids 39702 that constitute the linker (39306) in between SSM and `RBD’5 (Fig. 1a,b and Supplementary Fig. 1a). Two conformations have been observed at the Cterminal or `RBD’5 side on the linker, each hinged at L405 so that the CCR4 Formulation position of P404 wasNat Struct Mol Biol. Author manuscript; available in PMC 2014 July 14.Gleghorn et al.Pagevariable (Supplementary Fig. 2c). The observed variability raises the possibility that SSM could interact with `RBD’5 as a monomer (cis), dimer (trans), or both inside the crystal structure (Fig. 1b), but we cannot correlate either linker conformation using a monomeric or dimeric state. Each and every 649 interface is developed when the `V’-shape formed by SSM 1 and two straddles `RBD’5 1, while the `V’-shape produced by `RBD’5 1 and two straddles SSM 1 (Fig. 1b ). The intramolecular interactions of an SSM and an `RBD’5 type a core composed of residues with hydrophobic side chains (Fig. 1c). The external solvent boundary of this core is defined by Thr371 with the longer from the two SSM -helices, 1; Ser384 of SSM 2; Gln411, Tyr414, and Gln419 of `RBD’5 1; and Lys470 of `RBD’5 two (Fig. 1c). Each and every of these residues amphipathically contributes hydrophobic portions of their side chains for the core, with their polar element pointed outward. Val370, Ile374, Ala375, Leu378 and Leu379 of SSM 1 also contribute for the hydrophobic core as do Ala387, Ile390 and Leu391 of SSM two; `RBD’5 1 constituents Pro408 (which starts 1), Leu412, Leu415 and Val418; and Phe421 of L1 (Fig. 1c). Moreover, `RBD’5 two contributes Leu466, Leu469, Leu472 and Leu475 (Fig. 1c). On the two polar interactions in the SSM RBD’5 interface, one a basic charge is contributed by SSM Arg376: its two -amine groups hydrogen-bond with two carboxyl groups from the citrate anion present inside the crystal structure, while its – and -amines interact with the main-chain oxygens of, respectively, Glu474 and Ser473 which can be positioned close to the C-terminus of `RBD’5 two (Fig. 1d). SSM Arg376 is conserved in those vertebrates analyzed except for D. rerio, exactly where the residue is Asn, and Glu474 and Ser473 are invariant in vertebrates that contain the `RBD’5 2 C-terminus (Supplementary Fig. 1a). Inside the other polar interaction, the side-chain hydroxyl group of SSM Thr371 and also the main-chain oxygen of Lys367 hydrogen-bond using the amine group of `RBD’5 Gln419, when the -amine of Lys367 hydrogen-bonds using the hydroxyl group of Gln419 (Fig. 1c). SSM residues lacking strict conservation, i.e., Met373, Tyr380, Gly381, Thr383 and Pro385, are positioned on the solvent-exposed side, opposite for the interface that interacts with `RBD’5 (Supplementary Fig. 2d). Comparison of `RBD’5 to an RBD that binds dsRNA We had been surprised that the 3 RBD structures identified by the Dali server28 to be structurally most LTB4 Species similar to `RBD’5 do bind dsRNA (Supplementary Table 1). On the 3, Aquifex aeolicus RNase III RBD29 provides essentially the most comprehensive comparison. A structurebased sequence alignment of this RBD with hSTAU1 `RBD’5 revealed that even though the two structures are practically identical, hSTAU1 `RBD’5 features a slightly shorter loop (L)1, an altered L2, in addition to a longer L3 (Fig. 2a,b). Moreover, hSTAU1 `RBD’5 lacks crucial residues that typify the three RNA-binding Regions (Regions 1, two and 3) of canonical RBDs23 and that happen to be present within the A. aeolicus RNase III RBD (Fig. 2b). By far the most clear variations reside in Region 2.
